SAK3 confers neuroprotection in the neurodegeneration model of X-linked Dystonia-Parkinsonism

Abstract Background X-linked Dystonia-Parkinsonism(XDP) is an adult-onset neurodegenerative disorder that results in the loss of striatal medium spiny neurons (MSNs). XDP is associated with disease-specific mutations in and around the TAF1 gene. This study highlights the utility of directly reprogrammed MSNs from fibroblasts of affected XDP individuals as a platform that captures cellular and epigenetic phenotypes associated with XDP-related neurodegeneration. In addition, the current study demonstrates the neuroprotective effect of SAK3 currently tested in other neurodegenerative diseases. Methods XDP fibroblasts from three independent patients as well as age- and sex-matched control fibroblasts were used to generate MSNs by direct neuronal reprogramming using miRNA-9/9*-124 and thetranscription factors CTIP2 , DLX1 -P2A- DLX2 , and MYT1L . Neuronal death, DNA damage, and mitochondrial health assays were carried out to assess the neurodegenerative state of directly reprogrammed MSNs from XDP patients (XDP-MSNs). RNA sequencing and ATAC sequencing were performed to infer changes in the transcriptomic and chromatin landscapesof XDP-MSNs compared to those of control MSNs (Ctrl-MSNs). Results Our results show that XDP patient fibroblasts can be successfully reprogrammed into MSNs and XDP-MSNs display several degenerative phenotypes, including neuronal death, DNA damage, and mitochondrial dysfunction, compared to Ctrl-MSNs reprogrammed from age- and sex-matched control individuals’ fibroblasts. In addition, XDP-MSNs showed increased vulnerability to TNFα -toxicity compared to Ctrl-MSNs. To dissect the altered cellular state in XDP-MSNs, we conducted transcriptomic and chromatin accessibility analyses using RNA- and ATAC-seq. Our results indicate that pathways related to neuronal function, calcium signaling, and genes related to other neurodegenerative diseases are commonly altered in XDP-MSNs from multiple patients. Interestingly, we found that SAK3, a T-type calcium channel activator, that may have therapeutic values in other neurodegenerative disorders, protected XDP-MSNs from neuronal death. Notably, we found that SAK3-mediated alleviation of neurodegeneration in XDP-MSNs was accompanied by gene expression changes toward Ctrl-MSNs.


Background
X-linked dystonia-parkinsonism (XDP) is a Mendelian adult-onset neurodegenerative disorder that was initially identi ed in individuals from the island of Panay in the Philippines (1).The pathogenic gene lesion has been identi ed as a disease-speci c insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1 (2)(3)(4).The SVA contains a hexameric DNA repeat expansion (CCCTCT) n , the length of which is polymorphic among patients and inversely correlated to the age at disease onset (3,5).
The clinical disease manifestations of XDP are heterogeneous, with some individuals exhibiting initial dystonia that combines with or is replaced by parkinsonism over time versus others in which parkinsonian features are the predominant phenotype (6-8).The neuroanatomical substrates of these motor de cits have not been fully de ned, but neuroimaging studies in patients and neuropathological analyses of postmortem brain tissue have collectively shown that XDP involves progressive atrophy of the neostriatum that may be due primarily to the loss of medium spiny neurons (MSNs) (9)(10)(11)(12)(13)(14).However, the exact mechanisms and pathways underlying this degeneration remain largely unknown.
Here, we describe cellular phenotypes associated with XDP in MSNs directly reprogrammed from XDP patient-derived broblasts as a model system to understand the molecular underpinnings of MSN degeneration in XDP (14,15).Direct neuronal reprogramming offers several advantages to studying adult-onset pathological features since directly reprogrammed neurons mimic the chronological age of broblast donors as assessed by DNA-methylation-based epigenetic age contrasting neurons differentiated from induced pluripotent stem cells representing an embryonic state (16).Direct neuronal reprogramming method has been used to gain insights into adult-onset neurodegenerative states in other diseases, such as Huntington's disease and primary tauopathies (17)(18)(19)(20)(21)(22)(23).In addition, while XDP is strictly an inherited disorder, aging till contributes to the disease onset as in other neurodegenerative disorders (24) as XDP patients remain asymptomatic until they reach middle age.Cellular hallmarks of aging include changes in gene expression that are involved in DNA damage responses, synaptic transmission, mitochondrial function, insulin signaling, and lipid metabolism (16,24,25).In the modeling of adult-onset inherited neurodegenerative disorders such as Huntington's disease, these age-associated changes become exacerbated contributing to the onset of neurodegeneration (20,24,26,27).
In the current study, we used MSNs directly reprogrammed from XDP patient broblasts using MSNreprogramming effectors including the neurogenic microRNAs (miRNAs), miR-9/9*, and miR-124 (miR-9/9*-124), and striatum-enriched transcription factors CTIP2, DLX1-2 and MYT1L that guide the miRNAinduced conversion towards MSNs (17,18,28,29).Directly reprogrammed XDP-MSNs recapitulate neurodegenerative phenotypes that resemble features detected in the brains of XDP individuals and not present in MSNs reprogrammed from control adult broblasts.Interestingly, when comparing XDP vs. control broblasts, there were minimal differentially expressed genes (DEGs) but comparison of XDP vs control MSNs resulted in more than 500 signi cant DEGs that arose as a result of cells adopting the MSN identity.These DEGs were enriched for several genetic pathways including calcium dysregulation.We then tested whether SAK3, a T-type calcium channel agonist (30), would alleviate neurodegeneration in XDP-MSNs as SAK3 has been suggested to be neuroprotective in other neurodegenerative diseases such as Alzheimer's Disease and Lewy body dementia (30)(31)(32), and also shown to rescue pathology caused by Taf1 knockdown in rodent brains (33,34).We found that the neurodegeneration phenotype in XDP-MSNs could be mitigated by treatment with SAK3, which correlates with a signi cant portion of DEGs in XDP-MSNs reversing toward Ctrl-MSNs.Overall, our study shows that directly reprogrammed XDP-MSNs models the adult-onset neurodegeneration of XDP characterized by gene expression and chromatin dysregulation and demonstrate the protective effect of SAK3 in XDP patient-derived neurons.

Fibroblast lines
Adult dermal broblasts from symptomatic patients with XDP and healthy controls were acquired from the CCXDP and NINDS repositories (Supp Table 1) (4).Primary broblasts were cultured in broblast culture media (15% FBS in DMEM) until ready for reprogramming experiments.
Lentivirus was made by transfecting 293LE cells with 1.5 µg of pMD2.G, 4.5 µg of psPAX2, 6 µg of lentiviral plasmid, 600 µL of Opti-MEM and 48 µL of PEI solution.Individual plasmids were made into lentiviruses, but they were pooled together during concentration.Lentivirus supernatant was collected and ltered through a 0.45 µm PES membrane.A LentiX concentrator was added to the supernatant at a 1:4 ratio and left overnight at 4 ℃.The solution was spun down for 45 min at 1000 g and 4 ℃.The pellet was resuspended in 1/10th volume of PBS.7 ml of 10% sucrose solution was added to a high-speed centrifuge tube.The virus solution in PBS was carefully layered on top of the sucrose solution and centrifuged for 2 hours at 70,000 g at 4 ℃.The pellet was resuspended in 1/100th of the initial supernatant volume with 10% sucrose solution and frozen at -80 ℃ for future transduction.

Cell culture and reprogramming
Fibroblasts were rst expanded in broblast culture medium (15% FBS in DMEM).They were split into 6well plates for infection with lentivirus for the microRNAs and MSN-speci c transcription factors.Reprogramming was performed using the previously described method until postinduction day (PID) 25-35.Brie y, broblasts were cultured until con uent.They were then plated onto 6-well tissue culture plates for transduction.Transduction was performed on Day 0 with lentiviruses for 9/9*-124, rtTA, ctip2, dlx1-p2a-dlx2 and myt1l and 1000X Polybrene.The cells were spinfected for 30 minutes.On day 1, the cells were refed with broblast culture medium and 1 µg/ml doxycycline.On day 3, the same feeding was performed but with the addition of the antibiotics puromycin (3 mg/ml), blasticidin (3 µg/µl) and G418 (300 µg/µl).Reprogramming cells were then replated onto polyornithine-, bronectin-and laminin-coated coverslips on day 5. On day 6, blasticidin and G418 were added to the media for the second and last time.

Treatment with compounds
DNA damage rescue experiment in Fig.

Cell Death Assay
SYTODX-Green cell death assay (Thermo Fisher Scienti c) was conducted at postinduction days (PIDs) 0, 20, 25 and 30.SYTOX-Green was added to the cell medium at a concentration of 0.5 µM (1:10,000) with Hoechst (1:5000).Cells with Sytox and Hoechst were incubated in a 37 ℃ incubator for 20 minutes.Four uorescence images were taken at 10x per well of a 96-well plate using a GE InCell Analyzer 1000.Sytox-and Hoechst-positive cells were counted using InCell Analyzer software, and percentages of cell death were calculated using the number of SYTOX-Green/Hoescht cells.

RNA extraction and qPCR
Total RNA for qPCR was isolated using TRIzol (Invitrogen).Brie y, the cells were lysed using TRIzol at room temperature for 5 minutes.Chloroform (0.2 ml) was added per 1 ml TRIzol and shaken vigorously.
The solution was incubated at RT for 2-3 minutes followed by 15 min of centrifugation for 5 minutes at 12,000 g at 4°C.The aqueous phase was collected and transferred to a fresh microcentrifuge tube and mixed with 0.5 ml isopropanol per 1 ml TRIzol.Then, 0.5 µl of glycogen was added to the solution, followed by inversion of the tubes and incubation at -20°C for 24 hours.It was spun down for 10 minutes at 13,000 g at 4°C.The pellet was carefully washed with 1 ml 75% ethanol per 1 ml TRIzol.It was centrifuged for 5 minutes at 4°C at 13,000 g.The supernatant was carefully removed, and the pellet was resuspended in molecular grade water.The concentration was measured using a Qubit RNA quanti cation kit.
Equal amounts of RNA were used for cDNA.Reverse transcription was performed using SuperScript IV First Strand Synthesis SuperMix (Invitrogen, cat.# 18090050) according to the manufacturer's protocol.qPCR was performed with fast SYBR Green PCR Master Mix (Applied Biosystems, cat.# 4385612) using the StepOne Plus Real-Time PCR System (AB Applied Biosystems, Germany).All samples were run with three technical replicates.All ct values were rst normalized to the housekeeping gene HPRT1.The fold change was calculated based on the 2-ΔΔCT method.
RNA extraction for RNA sequencing was performed using the RNeasy Micro Kit (Qiagen, cat.# 74004) according to the manufacturer's protocol.Collected RNA samples were submitted to the Genome Access Technology Center at Washington University for library preparation and sequencing.

RNA sequencing and analysis
Samples were prepared according to library kit manufacturer's protocol, indexed, pooled, and sequenced on an Illumina NovaSeq 6000.Basecalls and demultiplexing were performed with Illumina's bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read.RNA-seq reads were then aligned to the Ensembl release 76 primary assembly with STAR version 2.5.1a(35).Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.6-p5(36).Isoform expression of known Ensembl transcripts were estimated with Salmon version 0.8.2 (37).Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected.The ribosomal fraction, known junction saturation, and read distribution over known gene models were quanti ed with RSeQC version 2.6.2 (38).All gene counts were then imported into the R/Bioconductor package EdgeR (39) and TMM normalization size factors were calculated to adjust for samples for differences in library size.Ribosomal genes and genes not expressed in the smallest group size minus samples greater than one count-per-million were excluded from further analysis.The TMM size factors and the matrix of counts were then imported into the R/Bioconductor package Limma (40).Weighted likelihoods based on the observed mean-variance relationship of every gene and sample were then calculated for all samples and the count matrix was transformed to moderated log 2 counts-per-million with Limma's voomWithQualityWeights (41).The performance of all genes was assessed with plots of the residual standard deviation of every gene to their average log-count with a robustly tted trend line of the residuals.Differential expression analysis was then performed to analyze for differences between conditions and the results were ltered for only those genes with Benjamini-Hochberg false-discovery rate adjusted p-values less than or equal to 0.05.
For each contrast extracted with Limma, global perturbations in known Gene Ontology (GO) terms, MSigDb, and KEGG pathways were detected using the R/Bioconductor package GAGE (42) to test for changes in expression of the reported log 2 fold-changes reported by Limma in each term versus the background log 2 fold-changes of all genes found outside the respective term.The R/Bioconductor package heatmap3 (43) was used to display heatmaps across groups of samples for each GO or MSigDb term with a Benjamini-Hochberg false-discovery rate adjusted p-value less than or equal to 0.05.Perturbed KEGG pathways where the observed log 2 fold-changes of genes within the term were signi cantly perturbed in a single-direction versus background or in any direction compared to other genes within a given term with p-values less than or equal to 0.05 were rendered as annotated KEGG graphs with the R/Bioconductor package Pathview (44).
To identify co-regulated gene networks, the Limma voomWithQualityWeights transformed log 2 countsper-million expression data was also analyzed by weighted gene correlation network analysis with the R/Bioconductor package WGCNA (45).Brie y, all genes were correlated across each other by Pearson correlations and clustered by expression similarity into unsigned modules using a power threshold empirically determined from the data.An eigengene was then created for each de novo cluster and its expression pro le was then correlated across all coe cients of the model matrix.Because these clusters of genes were created by expression pro le rather than known functional similarity, the clustered gene modules were given the names of random colors where grey is the only module that has any pre-existing de nition of containing genes that do not cluster well with others.These de-novo clustered genes were then tested for functional enrichment of known GO terms with hypergeometric tests available in the R/Bioconductor package clusterPro ler.Signi cant terms with Benjamini-Hochberg adjusted p-values less than 0.05 were then collapsed by similarity into clusterPro ler category network plots to display the most signi cant terms for each module of hub genes in order to interpolate the function of each signi cant module.The information for all clustered genes for each module were then combined with their respective statistical signi cance results from Limma to determine whether those features were also found to be signi cantly differentially expressed.
RNA sequencing analysis on SAK3-treated cells was performed using the same pipeline but with an updated version of the above programs.Total RNA integrity was determined using an Agilent Bioanalyzer or 4200 TapeStation.Library preparation was performed with 10 ng of total RNA with a Bioanalyzer RIN score greater than 8.0.ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per the manufacturer's protocol.cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, and cycles per burst 50 for 120 seconds.cDNA was blunt ended, an A base was added to the 3' ends, and then Illumina sequencing adapters were ligated to the ends.Ligated fragments were then ampli ed for 12-15 cycles using primers incorporating unique dual index tags.Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.The gene count le was used to further analyze the differentially expressed genes using RUVseq (RUVr) normalization and DEseq2 with a cutoff of logFC ≤-1 and ≥ 1 and adjusted P ≤ 0.05.We compared the upregulated genes and downregulated genes with DARs of ATAC-seq data (adjusted P ≤ 0.05, logFC ≤-1 and ≥ 1).The DEGs and DARs were used to further analyze gene ontology terms using the IPA program.
10. Leafcutter data processing and analysis BAM les from the RNA sequencing data were generated by STAR, using --outSAMstrandField intronMotif option (46).Samples were grouped by disease conditions and comparisons were done pairwise, as required for Leafcutter analysis.Differentially spliced clusters and introns were de ned by FDR < 0.05, and were exported using LeafViz (47).

ATAC sequencing and analysis
Omni-ATAC was performed as outlined in Corces et al. (48).In brief, XDP and Ctrl broblasts were reprogrammed as previously described until PID 30.On PID 30, MSNs were harvested using 0.25% trypsin and a cell scraper to collect the remaining cells.Each sample was treated with DNase for 30 minutes before collection.Approximately 50,000 cells were collected for library preparation.The transposition reaction was completed with Nextera Tn5 Transposase (Illumina Tagment DNA Enzyme and Buffer Kit, Illumina) for 30 minutes at 37 ℃, and library fragments were ampli ed under optimal ampli cation conditions.Final libraries were puri ed by the DNA Clean & Concentrator 5 Kit (Zymo).Libraries were sequenced on an Illumina NovaSeq S4 XP (Genome Technology Access Center at Washington University).
ATAC-seq analysis in directly reprogrammed neurons was performed as previously described ( 49) by using the ATAC-seq Integrative Analysis Package (AIAP) pipeline (50).The ATAC-seq FASTQ les were trimmed by Cutadapt and aligned to the reference genome by BWA.ATAC peaks were called by MACS and used to obtain read counts.Differential peaks were identi ed using RUVseq (RUVr) normalization and then DEseq2 with a cutoff of logFC ≤-1 and ≥ 1 and adjusted P ≤ 0.05.Gained peaks were de ned as open (more accessible) chromatin regions.Conversely, all reduced peaks were de ned as closed chromatin regions.We annotated genes nearest to open or closed regions using chipseeker and compared them with DEGs of RNA-seq data (adjusted P ≤ 0.05, logFC ≤ -1 and ≥ 1).
The reprogramming of MSNs was con rmed by RNA sequencing of starting broblasts and MSNs.RNA sequencing showed a battery of neuronal and MSN marker genes selectively enriched in reprogrammed MSNs (Fig. 1E).Fibroblast genes such as ITGB1 were successfully erased in reprogrammed MSNs, consistent with previous reports, while neuronal genes such as SNAP25 and PPP1R1B, an MSN marker, were enriched only in reprogrammed MSNs (Supp Fig. 1C).The expression of long genes de ned by genes more than 150 kb in length (from genomic START to STOP codons), a transcriptomic feature unique to neurons (51)(52)(53), was found to be signi cantly increased compared to broblasts, demonstrating the neuronal identity acquired in reprogrammed cells (Fig. 1F).
RNA sequencing validation was done by qPCR for neuronal and MSN marker gene expression.MAP2 and NEUN are pan-neuronal markers.nTAF1 is an isoform of TAF1 that is speci c to neurons (2,54).SRRM4 is a neuron-speci c splicing factor (55). PPP1R1B is a marker speci c to MSNs.These markers were signi cantly enriched similarly for both XDP and Ctrl reprogrammed MSNs compared to starting broblasts (Fig. 1G).As a measurement of neuronal identity, we assessed the membrane excitability of reprogrammed XDP-and Ctrl-MSNs by whole-cell recording.The reprogrammed MSNs showed voltagedependent inward currents as well as action potentials (APs) upon current injections.All cells patched red action potential (AP) with around 71% of cells ring multiple APs in Ctrl-MSNs (total # cells patched = 52) and around 76% cells ring multiple APs in XDP-MSNs (total # cells patched = 68) (Fig. 1H; right panel = representative APs and inward currents).Taken together, these results show that direct neuronal reprogramming by miR-9/9*-124 and CDM factors successfully generate MSNs from broblasts of XDP patients.

Patient-derived XDP-MSNs show spontaneous neurodegeneration
XDP is characterized by degeneration of MSNs in the striatum, therefore we assessed whether reprogrammed XDP-MSNs would exhibit similar neurodegenerative phenotypes as seen in patient brains.The SYTOX-Green assay, which marks cells undergoing cell death, indicated no signi cant difference between XDP and Ctrl broblasts postinduction day (PID) 0. However, as reprogramming cells acquire the identity of MSNs at PID 30 and beyond, XDP-MSNs from three independent XDP patients showed a signi cantly increased level of cell death compared to Ctrl-MSNs from three age-matched healthy individuals, as indicated by the number of SYTOX-Green signals over Hoescht signals (Fig. 2A).It is worth noting that XDP-MSNs underwent spontaneous cell death without adding extrinsic insults, suggesting that cell-intrinsic properties underlie the increased vulnerability to neurodegeneration in XDP-MSNs.A phenotype that accompanies neurodegeneration is DNA damage.As shown in Fig. 2B, we found that XDP-MSNs exhibited signi cantly increased levels of DNA damage, as marked by the positive staining of the DNA double-stranded break marker 53BP1.By quantifying the number of DNA breaks per cell, we found that XDP-MSNs contained a signi cantly higher number of DNA double-stranded breaks.8OHdG, a marker of oxidative damage, was also signi cantly increased in XDP-MSNs (Fig. 2C).To substantiate the relationship between cell death and DNA damage observed in XDP-MSNs, we tested whether blocking the cell death pathway induced by DNA damage would alter the cell death level in XDP-MSNs.Treating XDP-MSNs with KU60019, an inhibitor of ATM kinase that promotes cell death in response to DNA damage (18), rescued XDP-MSNs from cell death close to the control level (Fig. 2D), suggesting the interplay between DNA damage seen in XDP-MSNs and neuronal death.Altogether, we found that XDP-MSNs can capture cell-intrinsic properties underlying XDP-associated neurodegeneration.

Patient-derived XDP-MSNs show increased vulnerability to TNFα
We also tested whether XDP-MSNs would be hypersensitive to additional cellular insults.One such insult is TNFα, which is a pro-in ammatory molecule and can lead to cellular apoptosis (56).In XDP broblasts as well as XDP neuronal stem cells (NSCs), the NFκB pathway which is activated by TNFα has been shown to be mis regulated (57).We treated XDP-MSNs with TNFα to test whether TNFα would exacerbate neurodegeneration.We observed that when the cells were treated with increasing doses of TNFα, XDP-MSNs showed signi cantly increased cell death levels, while the same concentration of TNFα did not induce neuronal death in Ctrl-MSNs compared to the PBS control (Fig. 2E).These results suggest that XDP-MSNs showed selective sensitivity to TNFα compared to Ctrl-MSNs.We also assessed mitochondrial dysfunction as a degeneration-associated phenotype.Cytochrome c is a mitochondrial protein that translocates to the nucleus when cells have dysfunctional mitochondria and/or are undergoing apoptosis (58,59).Quanti cation of the percentage of cytochrome c-positive nuclei in Ctrl vs XDP-MSNs showed that XDP-MSNs treated with TNFα showed a signi cantly increased cytochrome c signal in the nucleus, whereas Ctrl-MSNs remained unaffected with or without TNFα treatment (Fig. 2F).Therefore, our results indicate that XDP-MSNs from multiple independent patients can reliably recapitulate increased susceptibility to cellular stress at doses that do not affect control cells.

Transcriptomic and chromatin landscape analysis of XDP-MSNs reveals dysregulation in pathways involved in neurodegenerative diseases
To gain further insights into the differential cellular state between XDP-MSNs and Ctrl-MSNs, we carried out comparative RNA-seq and ATAC-seq analyses to determine whether any potential differences in transcriptome and chromatin accessibilities would exist between XDP and Ctrl-MSNs (Fig. 3A, 4A).Both RNA and ATAC-seq were performed on PID 28.For RNA-seq analysis, differentially expressed genes (DEGs) that met the cutoff of logFC ± 1 with adj P-Val ≤ 0.05 were considered signi cant (Supp Table 3).The comparison between XDP-and Ctrl-MSNs resulted in 239 signi cantly upregulated and 463 signi cantly downregulated genes in XDP-MSNs (Fig. 3B).GO analysis was performed for the down or up regulated genes in XDP and that resulted in several terms associated with neuronal health such as "Regulation of response to stress", "Synaptic transmission, GABAergic" (Fig. 3C). Figure 3D shows representative IGV tracks of genes that are signi cantly downregulated (TDO2) or signi cantly upregulated (PAX3) in XDP-MSNs.
Further, weighted gene co-expression network analysis (WGCNA) was performed with the RNA-seq datasets to identify correlation between gene expression in XDP-MSNs with gene networks that cluster with those genes.WGCNA resulted in 5 modules that were signi cantly enriched in XDP-MSNs (p-value cutoff ≤ 0.05) compared to Ctrl-MSNs (Fig. 3E, Supp Table 4, 5).One of the signi cant modules was the "salmon" module with a positive correlation coe cient in the XDP-MSNs.This module included 919 genes (Supp Table 5) that were implicated in pathways such as splicing, autophagy, and mitophagy (Fig. 3G).Interestingly, the genes from this module were enriched for putative binding sites for several transcription factors with TAF1 being the most signi cant as analyzed by Enrichr (Fig. 3F).This is an interesting nding for XDP speci cally because the mutations associated with the disease are around TAF1 gene.TAF1 as the top enrichment TF is also observed in another study looking into proteomic analysis of XDP neuronal stem cells, or MSNs derived from induced pluripotent stem cells (60).
In addition, regulation of alternative splicing of genes have been implicated in neurodegenerative diseases as well as repeat expansion diseases (61).Previous studies have also shown that in XDP, the SVA insertion in intron 32 of the TAF1 gene leads to intron retention, suggesting that splicing alteration may occur in XDP (4, 62).To explore the alternative splicing differences in XDP-MSNs compared to Ctrl-MSNs, leafcutter analysis was performed which resulted in 30 genes that are signi cantly differentially splicing in XDP-MSNs (adjusted P ≤ 0.05) (Supp Table 6).Supp Fig. 2A shows representative splicing tracks in XDP-MSNs compared to Ctrl-MSNs.Among these genes, GTF2H2, GAS5, ARF4, RNF212 were detected top signi cant genes that showed alternative splicing differences in XDP compared to Ctrl-MSNs.Interestingly, KEGG pathway analysis shows that the genes that show alternative splicing in XDP-MSNs were implicated in other neurodegenerative diseases such as ALS, PD, HD and AD (Supp Fig. 2B).Notably, similar to the WGCNA results of the salmon module (Fig. 3), the differentially alternatively spliced genes were also enriched for putative TAF1 binding sites (Supp Fig. 2C).GO analysis reveals mRNA splicing and RNA binding as some of the most signi cant terms (Supp Fig. 2D-F).
In addition to transcriptome analyses, chromatin accessibility was measured by ATAC-sequencing (48) (Fig. 4A).Differentially accessible regions (DARs) that met the cutoff of logFC ± 1 with adj P-Val ≤ 0.05 were considered signi cant (Supp Table 7).XDP-MSNs had 1632 regions that were signi cantly open and 8040 regions that were signi cantly closed compared to Ctrl-MSNs.The heatmap in Fig. 4B shows open and closed regions that are within ± 2 kb of a gene transcription start site (TSS).Next, the signi cant regions/DARs (1632 open and 8040 closed) were annotated for genes within ± 2 kb of TSS that corresponded to that region using chipseeker.The results were ltered for duplicated genes (genes with multiple DARs) as well as the distal intergenic regions.This resulted in 4045 genes that had signi cant open or closed DARs in XDP-MSNs.The 4045 genes corresponding to the signi cant DARs were implicated in axonal guidance signaling and synaptogenesis among the pathways identi ed (Fig. 4C).
When we integrated the signi cant gene lists from RNA-seq and ATAC-seq datasets, 200 genes were upregulated or downregulated DEGs, containing opened or closed DARs (Fig. 4D).Examples of genes that are downregulated and closed (RNF212) or upregulated and open (DDX43) in XDP-MSNs are shown in Fig. 4E.Pathway analyses for these 200 genes were signi cantly enriched with functional pathways such as axonal guidance signaling and, more interestingly, calcium signaling (Fig. 4F).

SAK3, a T-type calcium channel enhancer, rescues XDP-MSN cell death
As shown in Fig. 4, molecular function analysis of DEGs and DARs in XDP-MSNs suggested dysfunction in neurodegeneration as well as calcium signaling pathways.We then further investigated the possibility of therapeutic compounds that have been evaluated in other neurodegenerative diseases to validate our transcriptomic ndings and to establish this model as a way to understand XDP disease.SAK3 is an activator of the T-type calcium channel (30).SAK3 has been shown to be bene cial in other neurodegenerative diseases, as well as in a TAF1-de cient animal model (32)(33)(34).Importantly, when XDP and Ctrl-MSNs were treated with SAK3, we observed that XDP-MSNs were signi cantly rescued from cell death in a dose-dependent manner compared to DMSO (Fig. 5A).This protective effect of SAK3 was detected in XDP-MSN lines from three independent XDP patients where the cell death of XDP-MSNs, initially approximately 40%, was reduced to a level similar to that of Ctrl-MSNs.Furthering this nding, we also measured the effect of SAK3 on DNA damage and mitochondrial dysfunction by quantifying DNA double stranded break with 53BP1 and cytochrome c in the nucleus, respectively.SAK3 led to reduction in the DNA damage level in XDP-MSNs (Fig. 5B).Also, SAK3 lowered cytochrome c translocation to the nucleus in XDP-MSNs suggesting improvement in mitochondrial integrity (Fig. 5C).Interestingly, the exacerbation of neurodegenerative phenotypes by TNFα as a cellular insult can be also resolved with the use of SAK3 in XDP-MSNs including the rescue of exacerbated cell death with SAK3 (Fig. 5D), and lowering of the cytochrome c translocation in the nucleus (Fig. 5E).Therefore, our results demonstrate the neuroprotective effect of SAK3 in patient-derived XDP-MSNs.
To further understand the mechanism of how SAK3 plays a role in rescuing cell death of XDP-MSNs, we performed transcriptomic analysis of XDP and Ctrl-MSNs treated with SAK3 or DMSO as a negative control.Interestingly, we found that when Ctrl-MSNs treated with SAK3 vs DMSO were compared, there were 3 signi cant DEGs, whereas XDP-MSNs treated with SAK3 vs DMSO resulted in 450 signi cant DEGs (Fig. 6A, Supp Table 8).Figure 6B shows representative IGV tracks of SNAP25, a neuronal gene that is not altered in XDP or with SAK3 treatment indicating the neuronal identity persists with SAK3 treatments, as well as an example of a gene PTAFR, a neurodegeneration and in ammation associated gene (63, 64) whose expression is upregulated in XDP but reduced by SAK3 treatment.EDN2, another gene that has been studied in the context of retinal degeneration (65), is downregulated in XDP-MSNs whereas SAK3 treatment increases its expression.Gene enrichment analysis of those 450 DEGs using Ingenuity Pathway Analysis revealed several pathways that were common with DEGs of SAK3 compared to DMSO.Synthesis of reactive oxygen synthesis was predicted to be downregulated with SAK3 (Fig. 6C) and the genes were also enriched for Ion homeostasis of cells (Fig. 6C).Furthermore, 55 genes that were differentially expressed in XDP-MSNs changed their expression directionality with SAK3 treatment toward the expression in Ctrl-MSNs (Fig. 6D).Gene enrichment analysis for these genes using KEGG and Gene Ontology analysis revealed pathways such as protein digestion and absorption, and cAMP signaling pathway (Fig. 6E).Taken together, these results indicate that the neuroprotective effect of SAK3 is characterized by gene expression changes including those DEGs in XDP-MSNs reversing towards healthy control MSNs.

Discussion
Over the past four decades, signi cant strides have been made in the eld of XDP research, shedding light on various aspects of the disorder, such as its pathogenesis, the role of TAF1 mutations, and transcriptomic differences, among others (66).However, despite these advancements, there has been no model system that accurately captures the haplotype, age of onset, and neurodegenerative changes observed in the brains of XDP patients.The present study represents a critical demonstration of modeling neurodegeneration of XDP using patient-derived neurons that enable investigations of the adult-onset cellular phenotypes of XDP in MSNs.To achieve this, a direct reprogramming method was utilized to convert XDP patient broblasts into MSNs, effectively recapitulating the genetic and chromatin dysregulation of XDP.In addition to the examination of XDP phenotypes, these reprogrammed XDP-MSNs can be harnessed to test therapeutic candidates that alleviate the neurodegeneration phenotypes.
We demonstrate that broblast samples are amenable to direct neuronal reprogramming approach results in the successful conversion of adult patient broblasts into mature MSNs, providing a robust model for studying XDP.XDP-MSNs exhibited signi cantly higher rates of cell death and phenotypes associated with neurodegeneration, including DNA damage and mitochondrial dysfunction.Previous research has indicated dysregulation in the DNA mismatch pathway genes in XDP (67), but direct evidence was shown in our study, as the increase in cell death observed in XDP-MSNs was found to be reversible through the blockade of the ATM/ATR kinase pathway, which plays a crucial role in DNA damage-induced cell death.Notably, our observations indicate a pronounced vulnerability of MSNs in XDP to cellular stress, particularly evidenced by a substantial increase in cell death when exposed to TNFα, an in ammatory cytokine known to activate the NFκB pathway.This nding is particularly signi cant, as the downregulation of the NFκB pathway and an increase in several proin ammatory markers have been identi ed as contributing factors in XDP pathogenesis (57,68).The dysregulation of NFκB as well as the involvement of TNFα has been studied in the context of other neurodegenerative diseases such as Parkinson's Disease (PD), where there is an enhancement in the levels of TNFα in the striatum (69).
Our results indicate signi cant transcriptomic and chromatin differences between XDP-MSNs and Ctrl-MSNs, with these differences arising as a consequence of the MSN cell fate.These chromatin and transcriptomic variations observed in XDP-MSNs are associated with several pathways that have connections to other neurodegenerative diseases, including the calcium signaling pathway (70).
Moreover, a recent study looking at aging in directly reprogrammed medium spiny neurons from longitudinally collected broblasts from healthy individuals identi ed dysregulation of the calcium signaling pathway (26).Therefore, restoring dysregulation of calcium signaling could be a concept that could be applied to age-associated onset of neurodegeneration.As such, the results of our study offer evidence that cellular pathways altered in XDP-MSNs can be restored by SAK3 to alleviate the neurodegeneration phenotype in XDP-MSNs.SAK3, a T-type calcium channel activator has previously been studied in the context of other neurodegenerative disorders and in a mouse model of Taf1 knockdown (31,33,34,(71)(72)(73).SAK3 has also been shown to rescue behavioral phenotypes in a rat model of Taf1 intellectual disability syndrome (33).Another study has indicated that the rescue of phenotype by SAK3 is due to improved proteosome activity that is reduced in AD (73).Encouragingly, treatment with SAK3 successfully ameliorated cell death and mitochondrial dysfunction in XDP-MSNs.Furthermore, we observed a distinct set of genes whose directionality changed in XDP-MSNs following SAK3 treatment, where the genes that respond to the SAK3 treatment have implications in neurodegeneration as implicated previous studies.For example, PTAFR is a proin ammatory gene involved in Alzheimer's disease (63) and is upregulated in XDP, but the expression level is reduced by SAK3 treatment.EDN2, a receptor that has been shown to be neuroprotective in retinal neurodegeneration (65) is downregulated in XDP-MSNs, while SAK3 treatment rescued the expression of EDN2.In addition, transcriptomic analysis with SAK3 treatment shows reduction of oxidative stress which is a validation of the phenotype rescue that was seen in SAK3 treated MSNs.These ndings re ect the protective effect of SAK3 in XDP-MSNs from phenotype to transcriptomic changes.

Conclusions
Our understanding of XDP's causal factors had been limited to prior discoveries highlighting variable expression of TAF1 isoforms (2,3,54,74), the pathogenic effect of SVA insertion (5,75), or DNA repair genes (67).However, our study has now validated the presence of additional genes that differ in XDP-MSNs and can be modulated by SAK3 that protects XDP-MSNs from neurodegeneration.Further investigations are warranted to elucidate the roles of genes altered in XDP-MSNs and how they may be targeted as potential therapeutic strategies for individuals affected by XDP.

Declarations
Ethics approval and consent to participate: Not applicable Consent for publication:

Figure 1 Medium
Figure 1